Diseases caused by kinetoplastid protozoa remain serious public health issues in many tropical and subtropical regions of the world. Recent work has revealed that these parasites exhibit novel mechanisms of gene expression and mRNA maturation. Clearly, these unique processes represent potential targets for chemotherapeutic intervention in treatment of the diseases caused by the kinetoplastids. The long term objective of this research project is to dissect the novel mechanisms of mRNA maturation in these orga- nisms. In the kinetoplastid protozoa, many protein coding genes are transcribed as long 'multicistronic' precursor mRNAs that are processed to maturity in a bi-molecular splicing event trans-splicing) in which a 39 nt spliced leader (SL) is ligated to the 5' terminus of each mature mRNA. The SL is transcribed as a short primary transcript-the SL-RNA-bearing the 39 nt SL at its 5' end. The SL-RNA is 5' capped prior to the trans-splicing event and therefore the cap on the mature mRNAs is derived from the cap on the SL-RNA. The 3' ends of mature mRNAs are defined by cleavage-polyadenylation events that are mediated by as yet undefined signals in the primary transcripts; no signal analogous to the 'AAUAAA' cleavage-polyadenylation signal of mammalian mRNAs has been identified. Experiments in this proposal are designed to elucidate the unique biochemical and molecular mechanisms of mRNA maturation in the kinetoplastid protozoa. Cell free nuclear extracts developed from T. cruzi and several other kinetoplastid protozoa are partially competent for several steps of mRNA processing; i.e., 5' capping, 3' polyadenylation, and several steps of trans-splicing. Using these extracts, another unique facet of mRNA maturation in the kinetoplastids has been discovered; i.e., the capping machinery in the nuclear extracts shows unprecedented specificity for certain RNAs. Thus, only the SL-RNA and the U-RNAs are capped in these extracts. Soluble capping enzymes from other eukaryotes show no specificity for any RNAs. This observation is particularly interesting in that the in vitro activity accurately reflects the in vivo capping requirements-since the mature mRNAs obtain their caps from the SL-RNA, the only RNAs that need be capped are the SL- and U-RNAs. Moreover, recent experiments have shown that the capping activity fractionates as a 15-20S RNA-protein complex on glycerol gradients or by large pore gel filtration chromatography. Experiments described herein are designed to identity the RNA primary sequence and/or secondary structure determinants of the specificity of the in vitro capping activity, and to identify, purify and characterize the trans-acting enzymes and factors in the capping complex.